One of the major problems of secretion is understanding the mechanisms whereby a secreted protein is translocated across the lipid bilayer. The energetic problems of this process are formidable. We are taking two approaches to the problem: 1) to define whether we can trap and isolate folding intermediates of secretory proteins in vitro to define whether they pass through hydrophobic states that may explain their interaction with and translocation across the membrane. Specifically, we are studying ovalbumin, the major secreted protein of chicken eggs. This protein was chosen for study because, unlike most secreted proteins, it is not synthesized as a precursor protein with an additional hydrophobic leader sequence. 2) We are also studying how to couple the in vitro synthesis of secreted proteins such as immunoglobulin and ovalbumin from mRNA to membranes using rough endoplasmic reticulum as an acceptor membrane. By using this approach, we hope to define the structures of the membrane that allow the recognition and translocation of secreted proteins.